This invention relates to a method and apparatus for the culturing of cells.
The culturing of living cells in vitro is desired for a variety of purposes such as the preparation of viral vaccines, the recovery of valuable by-products of the cell metabolism and the production of tissue-like densities for creating artificial organs.
Various procedures have been developed previously for the culturing of cells in vitro. One widely used method involves attaching and growing the cells on the interior surface of glass or plastic roller tubes and bottles. This method is exemplified by use of the Flow tube (Flow Laboratories) disclosed in U.S. Pat. No. 3,450,598. Another procedure used heretofore attaches and grows the cells on the flat side of appropriately shaped stationary containers such as, for example, the ordinary petri dish or rectangular shaped culture plates. The flat surface method also has been employed in apparatus having a stack of plates comprising a continuous plastic sheet arranged around a set of spaced-apart supports as illustrated in U.S. Pat. No. 3,843,454. Instead of using bare glass or plastic as the support surface for growing cells as monolayers, collagen-coated glass also has been employed. In order to provide a 3-dimensional support matrix for cell culturing, use of a collagen-coated cellulose sponge has been suggested heretofore.
Further background information on these and other such conventional cell culturing methods can be had by reference to a standard text in the field such as Kruse and Patterson, "Tissue Culture Methods and Applications," Academic Press, New York, 1973.
Recently, the use of hollow fibers or synthetic capillaries has been disclosed as a support matrix for the propagation of cells. This use was reported by Knazek, Science 178, 65-67 (1972), and specific apparatus for this cell culturing method is described in U.S. pat. Nos. 3,821,087 and 3,883,393. The apparatus comprises a bundle of ultrafiltration fibers retained in a cylindrical shell or cartridge. In essence, the apparatus employs membrane perfusion by artificial capillaries. The extensive surface area of the hollow fiber system allows selective transport through the fiber walls and facilitates molecular exchange between the stream flowing through the fiber interiors and a liquid which bathes the outer surfaces of the fibers by a simple gradient diffusion. This hollow fiber apparatus is further described by Knazek in Federation Proc. 33, 1978-81 (1974), and in Exptl. Cell Res. 84, 251-4 (1974) wherein it is used to produce HCG hormone from human choriocarcinoma cells at a rate eleven times higher than that grown on 75 cm..sup.2 monolayer flasks (Falcon). Cartridge apparatus of the type disclosed by Knazek is commercially available from Amicon Corporation and its use in cell culturing is described in American Lab. October 1974, pp. 33-38.
Notwithstanding the usefulness of the Knazek apparatus and method, it has been found in practice that employment of the bundle or cartridge configuration with fluid flow of culture medium through the elongate capillary membranes prevents complete penetration of the fiber bundle by the cells and sets up an undesirable gradient of medium flow. The inability of the cells to fully penetrate the fiber bundle results in uneven dispersion of the cells and incomplete utilization of the available fiber surface for cell attachment. The undesirable gradient consists of an uneven distribution and utilization of liquid culture medium. As the medium flows through the reactor, nutrients are more available to the cells near the inlet, and as the medium flows to the outlet, metabolic products such as lactic acid accumulate in the medium, thereby undesirably affecting pH and producing other toxic effects on the cells.